基于BCMA突变体构建BCMA CAR-T细胞体外杀伤功能评价模型

目的：为解决野生型B 细胞成熟抗原（BCMA）被γ 分泌酶切割导致表达不稳定的问题，构建抵抗γ 分泌酶切割的BCMA突变体并构建靶细胞，用于评价BCMA CAR-T 细胞的杀伤功能。方法：将野生型BCMA的穿膜域替换为人CD8α穿膜域，构建抵抗γ分泌酶切割的BCMA突变体（BCMA-CD8α TM），构建过表达该突变体的U266（U266BCMA Mut）、K562（K562BCMA Mut）、SKOV3（SKOV3BCMA Mut）和CHO（CHOBCMA Mut）细胞；构建装载NFAT-EGFP 报告基因的BCMA CAR Jurkat 细胞（BCMA-CAR-Jurkat-Reporter）与U266BCMA Mut 细胞共培养，采用FCM 检测该细胞中EGFP 表达水平以指示NFAT 激活水平，荧光素酶法检测BCMACAR-T 细胞对Luciferase 标记的K562BCMA Mut 细胞的杀伤作用，实时无标记动态细胞分析技术（RTCA）检测BCMA CAR-T 细胞对SKOV3BCMA Mut和CHOBCMA Mut细胞的杀伤作用。结果：应用γ分泌酶抑制剂LY411575 抑制γ分泌酶活性，显著增强野生型U266 细胞表面BCMA表达水平，平均荧光强度上调10倍以上；但撤除抑制剂后BCMA表达水平逐渐降低（P<0.01）；BCMA-CD8α TM突变体可抵抗γ 分泌酶的切割作用，在U266 细胞表面稳定表达（P>0.05）；U266 细胞及过表达BCMA-CD8α TM 的U266 细胞与BCMA-CAR-Jurkat-Reporter 细胞共培养后都可激活Reporter 系统、增强EGFP表达，但该效应在BCMA-CD8α TM过表达的U266细胞中更显著（P<0.01）；BCMA-CD8α TM在BCMA表达阴性的K562、SKOV3和CHO 3种靶细胞中成功过表达，且在LY411575处理下该突变体的表达水平仅有小幅度升高；荧光素酶法检测结果显示，不同效靶比下，BCMA CAR-T 细胞均可特异、高效杀伤过表达BCMA-CD8α TM的K562 细胞；RTCA 结果显示，不同效靶比下，BCMA CAR-T 细胞均可有效识别、杀伤过表达BCMA-CD8α TM的SKOV3和CHO细胞，但同等效靶比下的Mock-T 细胞无此效应。结论：本实验构建的BCMA-CD8α TM突变体能够抵抗γ分泌酶的切割，在多种靶细胞表面稳定表达，为评价BCMA CAR-T细胞体外杀伤的有效性和特异性提供多种检测手段。

Establishment of an in vitro cytotoxicity evaluation model for BCMA CAR-T cells based on BCMA mutants

Objective:To engineer a BCMA mutant resistant to γ-secretase cleavage in order to stabilize wild-type BCMA expression after γ-secretase cleavage and to generate target cells for measuring BCMA CAR-T cell cytotoxicity. Methods: Our study aimed to engineer a mutant BCMA protein (BCMA-CD8α TM) that could resist γ-secretase cleavage by replacing the transmembrane domain of the wild-type BCMA protein with the human CD8α sequence. Four different types of cells in which this mutant gene was expressed excessively were engineered, including U266 (U266BCMA Mut), K562 (K562BCMA Mut), SKOV3 (SKOV3BCMA Mut), and CHO (CHOBCMA Mut)cells. BCMA CAR Jurkat cells, loaded with the NFAT-EGFP reporter gene (BCMA-CAR-Jurkat-Reporter), were engineered and co- cultured with U266BCMA Mut cells. The expression level of EGFP was detected by FCM in order to indicate the activation level of NFAT.The cytotoxicity of BCMA CAR-T cells against Luciferase-labeled K562BCMA Mut cells was detected by the luciferase assay. Additionally,real-time cell analysis (RTCA) technique was employed to detect the cytotoxicity of BCMA CAR-T cells against SKOV3BCMA Mut and CHOBCMA Mut cells. Results: Application of γ -secretase inhibitor LY411575 to inhibit γ -secretase activity significantly enhanced the expression level of BCMA on the surface of wild-type U266 cells, and the average fluorescence intensity was increased by more than 10 times. However, the expression level of BCMA gradually decreased after removal of inhibitors (P<0.01). BCMA-CD8α TM mutant could resist the cleavage of γ -secretase and expressed stably on the surface of U266 cells (P>0.05). U266 cells and U266 cells overexpressing BCMA-CD8α TM were co-incubated with BCMA-CAR-Jurkat-Reporter cells, both of which could activate the Reporter system and enhance the expression of EGFP, but the effect was more significant in U266 cells overexpressing BCMA-CD8α TM (P<0.01). BCMA-CD8α TM mutants were successfully overexpressed in 3 BCMA-negative target cells, namely K562, SKOV3 and CHO cells, and the expression level of the mutant was only slightly increased under LY411575 treatment. Luciferase assay results showed that under different target-effect ratios BCMA CAR-T cells could all be specific and efficient in killing K562 cells overexpressing BCMA-CD8α TM. RTCA results showed that under different target ratios BCMA CAR-T cells could all effectively recognize and kill SKOV3 and CHO cells overexpressing BCMA-CD8α TM, but Mock-T cells with the same target ratio had no such effect. Conclusion: The BCMA-CD8α TM mutant engineered in this study can resist γ-secretase cleavage and exhibits stable surface expression on various target cells, thus offering various methods for evaluating the efficacy and specificity of BCMA CAR-T cell cytotoxicity in vitro.